The E3 ubiquitin ligase Mdm2 acts as a key factor in the degradation of p53 tumor suppressor in human cancer cells and inhibition of Mdm2 activity is a validated approach to activate p53 function in cancer therapy. Nevertheless, inhibitors of Mdm2 such as Nutlin-3 has many limitations, suggesting that additional targets in this pathway need to be further elucidated. The proposed studies aim to dissect the mechanisms of two newly identified ubiquitin ligases critically involved in ARF/p53-mediated tumor suppression and provide the unequivocal evidence for proof of concept to develop the inhibitors of these two potential critical targets in cancer therapy. ARF was originally identified as an alternative transcript of the Ink4a/ARF tumor suppressor locus. It is well accepted that ARF plays a major role in activating p53 function, specifically, in respond to oncogenic stress. Surprisingly, by usig a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, it has been found that the protection from tumorigenesis is absolutely dependent on ARF-mediated p53 activation in vivo. Thus, ARF apparently plays a much more important role in suppressing tumorigenesis in general, than originally anticipated. Restoration of the ARF-p53 function remains an important goal in the quest for more effective cancer therapeutics. Indeed, like ARF, Nutlin-3, a small molecule inhibitor of Mdm2, is able to activate p53, and exhibits antitumor efficacy in cancer cells that express wild-type p53. Nevertheless, although Nutlin-3 can effectively block the interaction of Mdm2 and p53, Nutlin-3 is also very effective at increasing the synthesis of Mdm2 mRNA and protecting Mdm2 from degradation. These effects of Nutlin-3 on Mdm2 lead to a rapid restore of cancer cell growth upon temporary removal of the compound, sabotaging the efficacy of nutlin-3 in cancer therapy. Thus additional cancer targets aiming at this pathway are clearly needed for more effective therapeutic purpose. Our preliminary studies reveal that targeting two novel ubiquitin ligases (ARF-BP1 and ULF) in the ARF-p53 pathway represents a particularly attractive approach to activate ARF-mediated tumor suppression in cancer therapy. The central hypothesis to be tested here is that inactivation of ARF-BP1 or ULF can effectively activate ARF-mediated function and suppress tumorigenesis in vivo. It includes the following two specific aims. In Aim 1, we will investigate whether inactivatin of ULF activity is sufficient to suppress tumorigenesis by using mouse models. In Aim 2, to validate whether ARF-BP1 is indeed a potential cancer target, we will use the ARF-BP1 mutant mice to examine whether inhibition of ARF-BP1 suppresses tumorigenesis in vivo in a manner reminiscent of ARF activation. PUBLIC HEALTH RELEVANCE: Restoration of the ARF-p53 function remains an important goal in the quest for more effective cancer therapeutics. Indeed, like ARF, Nutlin-3, a small molecule inhibitor of Mdm2, is able to activate p53, and exhibits antitumor efficacy in cancer cells that express wild-type p53. Nevertheless, the usage of Nutlin-3 has certain limitations. For example, although Nutlin-3 can effectively block the interaction of Mdm2 and p53, Nutlin-3 is also very effective at increasing the synthesis of Mdm2 mRNA and protecting Mdm2 from degradation. These effects of Nutlin-3 on Mdm2 lead to a rapid restore of cancer cell growth upon temporary removal of the compound, sabotaging the efficacy of nutlin-3 in cancer therapy. Thus additional cancer targets aiming at this pathway are clearly needed for more effective therapeutic purpose. The proposed studies aim to dissect the mechanisms of two newly identified ubiquitin ligases critically involved in ARF/p53- mediated tumor suppression and provide the unequivocal evidence for proof of concept to develop the inhibitors of these two potential critical targets in cancer therapy.